New application of gastrodia elata dizziness-relieving preparation

Abstract

The invention belongs to the field of traditional Chinese medicines, discloses a novel application of a gastrodia elata dizziness-relieving preparation, and particularly relates to an application of the gastrodia elata dizziness-relieving preparation in medicines for preventing and treating Parkinson's disease and motor complications thereof. The gastrodia elata dizziness relieving preparation is prepared from 12 raw medicinal materials including gastrodia elata, uncaria, rhizoma alismatis, pinellia ternate, bighead atractylodes rhizome, poria cocos, radix paeoniae alba, caulis bambusae in taeniam, ligusticum wallichii, liquorice, pericarpium citri reticulatae and ginger. Modern pharmacodynamic test research shows that the gastrodia elata dizziness relieving preparation can regulate expression of alpha-synuclein, LC3B and Beclin1 of a Parkinson's disease model rat group, and has a neuroprotective effect on the model rats; the gastrodia elata Jiangning granules have obvious effects of improving behavioristics of Parkinson's disease model mice, and have good prevention and treatment effects on motion fluctuation and dyskinesia of Parkinson's disease and complications of Parkinson's disease.

Description

Technical Field

[0001] The invention belongs to the technical field of traditional Chinese medicines, and specifically relates to a new use of a Tianma Xuanyunning preparation, in particular to the use of the Tianma Xuanyunning preparation in preparing a drug for treating Parkinson's disease. Background Art

[0002] The Chinese patent medicine Tianma Xuanyunning preparation that has been marketed includes two dosage forms, one is Tianma Xuanyunning granules (approval number: National Medicine Standard Z20060009), produced by Lunan Houpu Pharmaceutical Co., Ltd.; the other is Tianma Xuanyunning mixture (approval number: National Medicine Standard Z20083010), produced by Shaanxi University of Chinese Medicine Pharmaceutical Factory. Tianma Xuanyunning preparation is made of 12 raw materials: Tianma, Uncaria, Alisma, Pinellia, Atractylodes, Poria, White Peony, Rhizoma Anemarrhenae, Ligusticum chuanxiong, Licorice, Tangerine Peel, and Ginger. It has the effects of expectoration, relieving dizziness, and soothing the stomach and stopping vomiting. It is used for dizziness, nausea, vomiting, pale tongue, and white and slippery tongue coating, especially for Meniere's disease. At present, there is no report on the preventive and therapeutic effects of Tianma Xuanyunning preparation on Parkinson's disease.

[0003] Parkinson's disease (PD), also known as parkinsonism, is a common neurodegenerative disease in middle-aged and elderly people. It is characterized by progressive degeneration of dopaminergic neurons in the substantia nigra and the formation of Lewy bodies, as well as biochemical changes such as decreased dopamine transmitters in the striatum and imbalance of dopamine and acetylcholine transmitters. It manifests as motor symptoms such as tremor, muscle rigidity, bradykinesia, and postural balance disorders, as well as non-motor symptoms such as hyposmia, constipation, abnormal sleep behavior, and depression. The prevalence of Parkinson's disease increases with age, and the average age of onset is about 55-60 years old. With the aging of the population, its incidence rate is increasing year by year. It is a major disease that affects human health and life, and brings huge social and economic burdens to patients' families and society.

[0004] There is no consensus on the pathogenesis of Parkinson's disease. The main view is that it is related to abnormal protein aggregation, mitochondrial dysfunction, oxidative stress, etc. Clinically, the commonly used treatments for Parkinson's disease include drug therapy, mainly levodopa, surgical therapy, and physical therapy. However, at present, drugs are still the main treatment for Parkinson's disease at home and abroad, such as oral dopamine. However, adverse reactions such as muscle spasms, soreness, mental disorders, movement disorders, and leukopenia and thrombocytopenia often occur during medication.

[0005] Traditional Chinese medicine has certain efficacy and advantages in the treatment of Parkinson's disease. For example, when used in combination with Western medicine, it can improve the efficacy, reduce the adverse reactions of drugs, relieve the patient's motor and non-motor symptoms, improve the ability to live and work, improve the quality of life, and delay the progression of the disease. Traditional Chinese medicine has its own unique insights into the etiology and pathogenesis of Parkinson's disease, believing that the disease is located in the brain, attached to the tendons and veins, and is closely related to the dysfunction of the liver, kidney, spleen and other internal organs; the pathological nature is the deficiency of the root and the excess of the symptoms. The deficiency of the root is qi and blood deficiency, insufficient marrow, deficiency of yang qi, and deficiency of liver and kidney yin; the excess of the symptoms is wind yang movement, blood stasis, and phlegm turbidity.

[0006] However, different doctors have different understandings of the etiology and pathogenesis of Parkinson's disease and methods of syndrome differentiation and treatment. At present, it is clinically believed that the basic pathogenesis of Parkinson's disease is deficiency of the root and excess of the superficial symptoms. The deficiency is liver and kidney deficiency, qi and blood deficiency, and the excess is wind, fire, phlegm, and blood stasis. There are reports that Liuwei Dihuang Pills plus Chinese medicine paste formula nourishes the liver and kidney, extinguishes wind and calms tremors to treat liver and kidney yin deficiency type Parkinson's disease; Bazhen Decoction plus Chinese medicine paste is used to treat qi and blood deficiency type Parkinson's disease; Nanjing Traditional Chinese Medicine Hospital uses warm kidney and nourish liver formula to treat non-motor symptoms such as constipation and frequent urination caused by yang deficiency in Parkinson's patients; Guo Weiting uses Shaogan Dingchan Decoction to treat Parkinson's disease, nourish the kidney and blood, extinguish wind and stop tremors. At present, there is no clear Chinese patent medicine for the treatment of Parkinson's disease, so it is urgent to choose more traditional Chinese medicine to enrich the clinical treatment of Parkinson's disease. Summary of the invention

[0007] In order to enrich the drug options for clinical treatment of Parkinson's disease, the purpose of the present invention is to provide a new use of Tianma Xuanyunning preparation, especially the use of Tianma Xuanyunning preparation as a drug for preventing and treating Parkinson's disease and its motor complications.

[0008] Use of Tianma Xuanyunning preparation in drugs for preventing and treating Parkinson's disease and its motor complications.

[0009] The Parkinson's disease is of phlegm-heat-moving-wind type or phlegm-stasis-toxic-damage type.

[0010] The motor complications of Parkinson's disease are levodopa-induced.

[0011] In the present invention, the motor complications of Parkinson's disease include motor fluctuations and dyskinesias.

[0012] Dyskinesias include peak-dose dyskinesias, bidirectional dyskinesias, or dystonias.

[0013] The Tianma Xuanyunning preparation is made of Tianma, Uncaria rhynchophylla, Alisma orientalis, Pinellia, Atractylodes macrocephala, Poria, White Peony Root, Rhizoma Anemarrhenae, Ligusticum chuanxiong, Licorice, Tangerine peel and ginger.

[0014] The Tianma Xuanyunning preparation is in the form of granules, mixtures, pills, tablets, capsules, syrups and the like.

[0015] The Tianma Xuanyunning preparation is Tianma Xuanyunning granules or Tianma Xuanyunning mixture.

[0016] Pharmacodynamic studies have shown that Tianma Xuanyunning preparations can improve the hypoactivity, slow movements, poor balance and coordination of model mice, correct balance disorders in mice, extend the stride length of model mice, and alleviate the gait abnormalities of Parkinson's model mice. Tianma Xuanyunning granules can significantly improve the behavior of Parkinson's disease model mice and have a positive therapeutic effect on motor fluctuations and dyskinesias in Parkinson's disease and its complications.

[0017] Tianma Xuanyunning preparation can regulate the expression of α-synuclein, LC3B, and Beclin1 in Parkinson's disease model rats, and may play a neuroprotective role by upregulating the autophagic activity of striatal neurons and clearing excess α-synuclein. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 The effect of Tianma Xuanyunning granules on the climbing time of Parkinson's disease model mice in the pole climbing experiment. Note: Compared with the blank control group, " # "P<0.001, compared with the model control group," * "P<0.05," *** ” indicates P<0.001.

[0019] Figure 2 This is the step length experiment of Tianma Xuanyunning Granules on the step length of Parkinson's disease model mice. Note: Compared with the blank control group, " # "P<0.05, compared with the model control group," * ” indicates P<0.05.

[0020] Figure 3 The swimming experiment is the effect of Tianma Xuanyunning Granule on the swimming score of Parkinson's disease model mice. Note: Compared with the blank control group, " # "P<0.001, compared with the model control group," * ” indicates P<0.001.

[0021] Figure 4 The effect of Tianma Xuanyunning Granules on the number of climbing grids of Parkinson's disease model mice in the mine experiment. Note: Compared with the blank control group, " # "P<0.001, compared with the model control group," * " indicates P < 0.05," ** ” indicates P<0.01.

[0022] Figure 5 The effect of Tianma Xuanyunning Granules on the number of upright standing times of Parkinson's disease model mice in the mine experiment. Note: Compared with the blank control group, " #"P<0.001, compared with the model control group," * " indicates P < 0.05," *** ” indicates P<0.001. DETAILED DESCRIPTION

[0023] According to the above contents of the present invention, in accordance with common technical knowledge and customary means in the art, without departing from the above basic technical ideas of the present invention, other various forms of modification, replacement or change may be made.

[0024] The above contents of the present invention are further described in detail below through specific implementation methods in the form of embodiments. However, this should not be understood as the scope of the above subject matter of the present invention being limited to the following examples. All technologies realized based on the above contents of the present invention belong to the scope of the present invention.

[0025] Experimental Example 1 Study on the effect of Tianma Xuanyunning granules on the behavior of MPTP-induced Parkinson's disease model mice

[0026] 1 Experimental Materials

[0027] 1.1 Experimental animals

[0028] Sixty healthy and clean adult male C57BL / 6 mice, weighing 20±2 g, were provided by Lunan Pharmaceutical Group Co., Ltd., with experimental animal license: SYXK(Lu)20230023. They were raised in the Pharmacology Center of Lunan Pharmaceutical Group. All mice were adaptively raised for 1 week in a breeding environment at 22-26°C, with free food, water, and adequate ventilation.

[0029] 1.2 Reagents, drugs and instruments

[0030] 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP for short), Shanghai Yuanye Biotechnology Co., Ltd.; Tianma Xuanyunning Granules (approval number: National Medicine Standard Z20060009), Lunan Houpu Pharmaceutical Co., Ltd. BS223S electronic balance, Sartorius Scientific Instruments (Beijing) Co., Ltd.; 100cm×100cm×50cm mine box, homemade.

[0031] 2 Experimental methods

[0032] 2.1 Animal grouping, modeling and drug administration

[0033] Sixty male C57BL / 6 mice were randomly divided into three groups: blank control group, model control group, and Tianma Xuanyunning granule group, with 20 mice in each group, and they were housed in separate cages.

[0034] The Parkinson's disease model was induced by intraperitoneal injection of MPTP. The blank control group was intraperitoneally injected with an equal volume of normal saline, and the model control group and Tianma Xuanyunning granule group were intraperitoneally injected with 0.3 ml of 0.3% MPTP solution prepared with normal saline for 7 consecutive days (30 mg / kd·d -1 ), each time with an interval of 24 hours, to create a subacute Parkinson's disease mouse model, and observe the behavioral changes of the modeled mice. If symptoms such as limb tremor, gait incoordination, bradykinesia, and tail rigidity appear, the model is considered to be successful.

[0035] The Tianma Xuanyunning Granule group was intragastrically administered with 3.2 g / kg of Tianma Xuanyunning Granules daily, and the blank control group and the model control group were intragastrically administered with an equal volume of distilled water daily for 14 consecutive days.

[0036] 2.2 Experimental process

[0037] 2.2.1 Behavioral Experimental Procedure

[0038] Before modeling, mice were trained to perform pole climbing test, swimming test and mine field test, and mice with uncoordinated gait were eliminated. The above three tests and gait analysis test were performed at 10 am on the 0th day (before administration), 3rd day, 7th day and 14th day of the experiment, and the experimental data were recorded.

[0039] 2.2.1.1 Pole climbing experiment

[0040] A 2cm diameter plastic ball was fixed on a 50cm high iron frame. A layer of medical tape was spirally pasted on the iron rod to prevent slipping. The mouse placed its hind legs on the top plastic ball, head down, and its front paws grasped the iron ring to climb down naturally. The mouse's climbing behavior was observed and the climbing time was recorded. The climbing process was completed when the mouse's hind legs stepped on the iron frame platform at the bottom of the rod. Each mouse repeated 3 times and the average value was taken. If the mouse paused halfway or turned around to climb to the top of the rod, the test was invalid and needed to be repeated.

[0041] 2.2.1.2 Gait analysis

[0042] Prepare a piece of white paper about 1m long, and use foam boards to make crawling corridors every 10cm. There are three groups of 10 mice in each group, and a total of 6 sets of corridors. Dip the front paws of the mice in ink and put them in the corridors to guide them to move forward. Excluding the first 7cm and the last 7cm steps, observe whether the gait of the animal model is stable. Observe and record the distance between 5 consecutive front paws (the longest 5) and take the average value.

[0043] 2.2.1.3 Swimming test

[0044] Take a 3000mL transparent glass beaker, pour water at a temperature of 26℃ to 30℃ into the beaker, put the mice in the water to swim, and record the mouse swimming score. Scoring criteria: 3 points for continuous swimming, 2.5 points for swimming most of the time with occasional floating, 2 points for floating 50% of the time, 1.5 points for floating time significantly longer than swimming time, 1 point for floating on one side of the beaker and occasionally paddling with the hind legs, and 0 points for swimming time of 30s < 90s. The recording time is 5 minutes.

[0045] 2.2.1.4 Mine experiment

[0046] A 100cm×100cm×50cm mine box was made by ourselves, with the bottom divided into 16 squares of 4×4 evenly, and the top was not capped. A 60-watt light bulb was hung about 20cm above the mine box for lighting. The observation environment was a quiet darkroom, and the observation indicators were two: the number of grids climbed, recording the number of squares climbed by the mouse's limbs, which was the horizontal movement frequency; the second was the number of uprights, when the mouse stood on its hind legs and the front paws were more than 1cm off the ground, which was the vertical movement frequency; the number of uprights did not include the number of times the mouse groomed itself upright. The observation time for each mouse was 5 minutes.

[0047] 2.2.2 Observation of behavioral indicators

[0048] The behavioral observation time of mice was a few minutes and a few hours after modeling every day. On the 0th, 3rd, 7th and 14th days of oral administration, four behavioral experiments including pole climbing test, mine field test, swimming test and gait analysis were performed on the mice to observe the behavioral changes before and after modeling, and the experimental data were recorded and analyzed.

[0049] 2.2.3 Data Analysis Methods

[0050] SPSS 26.0 software was used for statistical analysis, and the measured data were expressed as mean ± standard deviation. The independent sample t-test was used to analyze the differences between the components, and P < 0.05 was considered statistically significant.

[0051] 3 Experimental results

[0052] 3.1 General Observations

[0053] The mice in the modeling group showed varying degrees of tremor, erect hair, and tail lift 10 to 20 minutes after the first injection of MPTP. The above symptoms gradually alleviated after 4-5 hours, but there were still changes such as reduced activity and slow movements. After 7 consecutive injections, the mice in the model control group showed symptoms such as staggering gait, reduced activity, and slowed movements. After intragastric administration of Tianma Xuanyunning Granules, the above symptoms were significantly alleviated, and behavioral activities improved. The blank control group did not show the above behavioral changes.

[0054] 3.2 Pole climbing experiment results

[0055] The coordination and balance ability of mice and bradykinesia can be evaluated by the pole climbing test. The shorter the time it takes for mice to climb the pole, the stronger the attachment ability of their limbs and the better the coordination; the longer the time it takes for mice to climb the pole, or the more they stop climbing, the worse the coordination and the weaker the attachment ability. Figure 1 . Figure 1 The results showed that before administration, the climbing time of the model group mice was significantly longer than that of the blank group mice (P < 0.05); the climbing time of the experimental mice in the Tianma Xuanyunning granule group was reduced compared with the model control group mice on the third day of administration. With the extension of the administration time, the climbing time of the Tianma Xuanyunning granule group mice was significantly shortened compared with the model control group mice on the 7th and 14th days of the experiment, with significant differences (P < 0.05, P < 0.001). This shows that the model mice can improve their limb movement slowness and balance coordination after being administered Tianma Xuanyunning granules.

[0056] 3.3 Gait analysis results

[0057] Gait analysis mainly evaluates the gait abnormality of the mouse's limbs. The shorter the average step length between the mouse's front paws, the more abnormal the mouse's gait. Figure 2 .from Figure 2 It can be seen that before administration, the average stride length of mice in the modeling group was significantly shortened compared with the blank control group and the modeling group (P < 0.05); compared with the model control group and the Tianma Xuanyunning Granule group, the average stride length of mice in the Tianma Xuanyunning Granule group increased on the 3rd and 7th days of the experiment. With the increase in the number of days of oral administration, on the 14th day of the experiment, the average stride length of mice in the Tianma Xuanyunning Granule group increased significantly compared with the model control group, and the difference was statistically significant (P < 0.05). This shows that Tianma Xuanyunning Granule can alleviate the problem of abnormal gait in Parkinson's model mice, which is to prolong the average stride length.

[0058] 3.4 Swimming test results

[0059] The lower the score of the mouse in the swimming test, the longer the mouse floats, and the more severe the behavioral abnormalities such as tremor and muscle rigidity of the mouse. This experiment can reflect the mouse's limb movement coordination ability. Figure 3 .from Figure 3It can be seen that before administration, compared with the blank control group and the modeling group, the blank control group mice mainly swam continuously, while the modeling group mice floated for a significantly longer time, and tremors, tilting performance of imbalanced balance and tail rigidity in the two groups of mice were observed, with significant differences between the two groups (P < 0.001); the swimming time of the mice in the Tianma Xuanyunning granule group was significantly prolonged compared with the mice in the model control group on the 7th and 14th days of the experiment, and the difference was extremely significant (P < 0.001). This shows that Tianma Xuanyunning granules can reduce the tremor amplitude of mice, significantly prolong the swimming time, and significantly enhance the balance and coordination ability. It shows that Tianma Xuanyunning granules can promote the recovery of the limb movement coordination ability of mice and play a role in correcting the balance disorder of mice.

[0060] 3.5 Mine Experiment Results

[0061] The mine experiment was conducted in a quiet, dark room. It mainly observed the spontaneous movement ability of mice within 5 minutes. The observation indicators were the number of horizontal climbing and the number of upright standing, which are the main indicators used to detect the lack of movement of mice after MPTP-damaged neurons. Figure 4 and 5 . The results showed that before administration, there was a significant difference in the number of crawling between the blank control group and the model group (P < 0.001), indicating that the number of crawling of mice in the model group was significantly reduced; 3 days after administration, there was no statistical difference between the model control group and the Tianma Xuanyunning group (P > 0.05); 7 and 14 days after oral administration, there was a significant difference in the number of crawling between the Tianma Xuanyunning granule group and the model control group (P < 0.05, P < 0.01). In terms of the number of upright standing, there was a significant difference between the model group and the blank control group (P < 0.001), indicating that the model was successfully established, and the spontaneous upright movement of mice in the model group was significantly reduced, indicating that MPTP damaged the dopaminergic neurons of mice. After 3 days of administration, the number of upright movements of mice in the Tianma Xuanyunning Granule group increased compared with the model control group and the Tianma Xuanyunning Granule group. After 7 and 14 days of continuous administration, the number of upright movements of the Tianma Xuanyunning Granule group increased significantly compared with the model control group, and the difference was statistically significant (P < 0.05, P < 0.001). This shows that Tianma Xuanyunning Granule has a significant improvement effect on the phenomenon of hypoactivity in mice.

[0062] The above experiments show that Tianma Xuanyunning Granules can significantly improve the behavior of Parkinson's disease model mice, and treat the motor fluctuations and dyskinesias of Parkinson's disease and its complications.

[0063] Experimental Example 2 Effect of Tianma Xuanyunning Granules on Parkinson's Disease Model Rats Induced by 6-OHDA

[0064] 1 Experimental Materials

[0065] 1.1 Experimental animals

[0066] Sixty healthy and clean adult male SD rats, 8 weeks old, weighing 200±20g, were provided by Lunan Pharmaceutical Group Co., Ltd., with experimental animal license: SYXK(Lu)20230023. They were raised in the Pharmacology Center of Lunan Pharmaceutical Group. All rats were adaptively cultured with ordinary feed for 1 week, exposed to natural light, at room temperature of 22-24℃, with free access to water and food.

[0067] 1.2 Reagents, drugs and instruments

[0068] 6-hydroxydopamine (6-OHDA), Sigma, USA; sodium pentobarbital, Wuhan Jinnuo Chemical Co., Ltd.; apomorphine (APO), Cayman, USA; α-synuclein antibody, LC3B antibody, Beclin1 antibody, Biosun, USA; normal goat serum, Shanghai Xinyu Biotechnology Co., Ltd.; dopamine tablets, national medicine standard H10930198, Shanghai Roche Pharmaceuticals Co., Ltd.; Tianma Xuanyunning granules, national medicine standard Z20060009, Lunan Houpu Pharmaceutical Co., Ltd. Multifunctional brain stereotaxic instrument, Stoelting, USA; FV3000 laser confocal microscope, Qlympus, Japan; Plus 384 microplate reader, Bio-Rad, USA.

[0069] 2 Experimental methods

[0070] 2.1 Animal grouping, modeling and drug administration

[0071] Sixty rats were randomly divided into 6 groups, with 10 rats in each group, namely blank control group, model control group, western medicine control group, and Tianma Xuanyunning Granule low-, medium-, and high-dose groups.

[0072] Except for the blank control group, the rest of the rats were modeled as follows. The rats were fixed on a stereotaxic apparatus, the hair on the top of the skull was cut off, and the rats were disinfected with iodine. The skin on the top of the skull was cut along the midline, the periosteum was peeled off, and the anterior and posterior bregma were exposed. The surface of the skull was wiped with hydrogen peroxide, and the bregma was used as the reference. According to the "Rat Brain Stereotaxic Atlas", the two coordinates of the right substantia nigra were determined: (1) 5.2mm behind the bregma, 1.5mm to the right of the midline, and 9.0mm below the dura mater; (2) 5.2mm behind the bregma, 2.5mm to the right of the midline, and 8.6mm below the dura mater. The skull was carefully drilled at the required surgical site with a dental drill, and a 5μl microinjector was used to slowly inject 6-OHDA (dissolved in saline containing 0.02% ascorbic acid, with a concentration of 2μg / μl) solution into the right substantia nigra, 3μl per hole. After the injection, the needle was retained for 5min and the needle was slowly withdrawn. After the operation, the skull hole was filled with medical gelatin sponge, the incision skin was sutured, and 200,000 units of gentamicin were injected intramuscularly for 7 days. After the rats woke up, they were returned to the cage for breeding. The blank control group rats were only injected with an equal amount of saline containing 0.02% ascorbic acid, and the other conditions were the same as the modeling operation. Ten days after the modeling operation, the rats were induced to rotate to the healthy side by intraperitoneal injection of about 0.5 mg / kg of apomorphine APO, and the number of rotations from the beginning to 30 minutes was recorded. The rats with an average number of rotations of more than 7 times per minute were considered to be successful models.

[0073] After successful modeling, rats in the western medicine control group were gavaged with 75 mg / (kg·d) of dopamine tablets, and rats in the low-, medium-, and high-dose groups of Tianma Xuanyunning Granules were gavaged with 1.08 g / (kg·d), 2.16 g / (kg·d), and 4.32 g / (kg·d) of Tianma Xuanyunning Granules, respectively. The blank control group and the model control group were gavaged with an equal volume of normal saline, and the drugs were given continuously for 8 weeks.

[0074] 2.2 Experimental observation indicators and methods

[0075] 2.2.1 Observation of general condition of rats

[0076] During the experiment, the rats' fur, spirit, activity, limb tremor, rigidity, etc. were observed daily.

[0077] 2.2.2 Morphological observation of striatum

[0078] After the experiment, the rats were killed, the brains were removed, the striatum was separated, and fixed by immersion in 4% formaldehyde. After 48 hours, the brains were rinsed in tap water for 30 minutes, dehydrated, transparent, and paraffin-embedded. Serial coronal sections were made, and HE staining was used to observe the morphological changes of striatal neurons in each group of rats under a microscope.

[0079] 2.2.3 Expression of α-synuclein antibody, LC3B antibody, and Beclin1 antibody in the striatum

[0080] Immunohistochemistry was performed according to the instructions of the kit, counterstained with hematoxylin, sealed, and observed under a microscope. Three striatal jar sections were taken from each rat, and four non-overlapping fields were randomly selected from each section. The average gray value of each field was obtained by the automatic image analysis system, and the average gray value of the four fields was averaged.

[0081] 2.2.4 Statistical methods

[0082] SPSS 26.0 software was used to process and statistically analyze all experimental data, and the data were expressed as “mean ± standard deviation”. When comparing data, if they conform to normal distribution, the variance homogeneity is tested. If the variance is homogeneous, one-way analysis of variance is used, and P < 0.05 is considered statistically significant.

[0083] 3 Experimental results

[0084] 3.1 Observation of general condition of rats in each group

[0085] The rats in the blank control group had shiny fur, good spirits, normal activities and eating and drinking, and no limb tremor or rigidity. The rats in the model control group had dull fur, obvious hair loss, frequent limb tremor and rigidity, and reduced drinking and food intake. The rats in the low-dose and medium-dose groups of Tianma Xuanyunning had dull fur, hair loss, erect hair, limb tremor, rigidity, and slow movement; and reduced food intake. The rats in the western medicine control group and the high-dose group of Tianma Xuanyunning granules had dull fur, some hair loss, trembling forepaws, slow movement, and slow limb extension. The symptoms and food intake gradually alleviated with the extension of medication time.

[0086] 3.2 Morphological changes of striatum in rats of each group

[0087] The neurons in the striatum of the rats in the blank control group were dense, normal in morphology, and neatly arranged. The neurons in the rats in the model control group and the low-dose group of Tianma Xuanyunning Granules were severely damaged, with a significant decrease in the number of neurons, a reduction in cell volume, wrinkled cell membranes, deformed nuclei, and wrinkled and marginalized nuclear chromatin. The striatal cell morphology of the rats in the western medicine control group and the medium- and high-dose groups of Tianma Xuanyunning Granules was greatly improved compared with the model control group.

[0088] 3.3 Expression of α-synuclein, LC3B, and Beclin1 in the striatum of rats in each group

[0089] Modern research shows that the main pathological characteristics of Parkinson's disease are the progressive reduction of dopaminergic neurons in the compact part of the substantia nigra in the midbrain and the appearance of Lewy bodies in neurons. α-synuclein is the main protein that constitutes Lewy bodies. The misfolding and aggregation of α-synuclein are the central events in the development of Parkinson's disease. The two main pathways for the body to repair or eliminate abnormal proteins are the ubiquitin proteasome system and the autophagy lysosome pathway. Both pathways can eliminate misfolded and aggregated proteins in the body by degradation. The important pathway for the degradation of oligomers and aggregates of α-synuclein that has been determined is the autophagy lysosome pathway. Studies have shown that the dysfunction of the autophagy lysosome pathway in nerve cells is closely related to the formation of Lewy bodies. Autophagy is a protein degradation method shared by eukaryotic cells that maintains normal cell function by degrading excessive or abnormal proteins, organelles, etc. in the cell. The regulatory mechanism of autophagy is very complex, and many proteins including LC3B and Beclin1 are involved. Therefore, it is of great significance to determine the expression of α-synuclein, LC3B, and Beclin1 in the striatum.

[0090] α-Synuclein, LC3B, and Beclin1 were mainly expressed in the cytoplasm of neurons and the processes of glial cells, and were expressed as brown-yellow coarse granules. The expression of α-synuclein, LC3B, and Beclin1 in the striatum of rats in each group is shown in Table 1. As shown in Table 1, the morphology of striatal neurons in rats in the blank control group was normal, and almost no α-synuclein deposition was observed, while the expression of LC3B and Beclin1 was high. Compared with the blank control group, the gray value of α-synuclein in the model control group, the western medicine control group, and the low, medium, and high dose groups of Tianma Xuanyunning Granules was significantly decreased, and the gray value of LC3B and Beclin1 was significantly increased (P < 0.05). Compared with the model control group, the gray value of α-synuclein in rats in the western medicine control group and the medium and high dose groups of Tianma Xuanyunning Granules was increased, and the gray value of LC3B and Beclin1 was significantly decreased (P < 0.05).

[0091] Table 1 Comparison of grayscale values ​​of α-synuclein, LC3B and Beclin1 expression in the striatum of rats in each group

[0092]

[0093] Note: Compared with the blank control group, # "P<0.01," ## ” indicates P<0.001; compared with the model control group, “ * " indicates P < 0.05," ** ” indicates P<0.01.

[0094] The above experiments show that after intervention with the western medicine dopamine tablets or the traditional Chinese medicine Tianma Xuanyunning granules, the striatal morphology of rats was improved compared with the model group, the expression of α-synuclein decreased, and the expression of LC3B and Beclin1 increased, indicating that the decreased autophagic activity of striatal neurons in Parkinson's disease model rats is related to the deposition of α-synuclein. Tianma Xuanyunning granules may play a neuroprotective role by upregulating autophagic activity and clearing excess α-synuclein.

Claims

1. Use of Tianma Xuanyunning preparation in the prevention and treatment of Parkinson's disease and its motor complications.

2. The use according to claim 1, characterized in that The Parkinson's disease is of phlegm-heat-moving-wind type or phlegm-stasis-toxic-damage type.

3. The use according to claim 1, characterized in that The motor complications of Parkinson's disease are levodopa-induced.

4. The use according to claim 1, characterized in that The motor complications of Parkinson's disease include motor fluctuations and / or dyskinesias.

5. The use according to claim 4, characterized in that The dyskinesias include peak-dose dyskinesias, bidirectional dyskinesias and / or dystonias.

6. The use according to claim 1, characterized in that: The Tianma Xuanyunning preparation is made of Tianma, Uncaria rhynchophylla, Alisma orientalis, Pinellia, Atractylodes macrocephala, Poria, White peony root, Rhizoma Anemarrhenae, Ligusticum chuanxiong, Licorice, Tangerine peel and ginger.

7. The use according to claim 6, characterized in that The Tianma Xuanyunning preparation is in the form of granules, mixtures, pills, tablets, capsules or syrups.

8. The use according to claim 7, characterized in that The Tianma Xuanyunning preparation is a mixture or granules.

9. The use according to claim 7, characterized in that: The Tianma Xuanyunning preparation is Tianma Xuanyunning granules or Tianma Xuanyunning mixture.

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